Transcript PowerPoint Presentation from June

Protein Identification
and Peptide Sequencing
by Liquid Chromatography –
Mass Spectrometry
Detlef Schumann, PhD
Director, Proteomics Laboratory
Department of Genome Science
May 27, 2005
The Proteomics Problem
Cell state 1
Protein 1
- Protein name: ...
- MW: ...
- Amino acid sequence: ...
- Modifications: ...
Why are
state 1 and 2
different?
Cell state 2
Proteomics
Protein 2
-Protein name: ...
- MW: ...
- Amino acid sequence: ...
- Modifications: ...
•
•
•
The Typical Proteomics Problem
Sample #488
Sample #487
MW
200
10
4
7
pI
4
7
pI
The Proteomics Laboratory at the GRI
Electrophoresis Laboratory
• 1-D gel electrophoresis (small format)
• 2-D gel electrophoresis (small and large format)
• Silver staining and Coomassie staining
• Imaging densitometry of protein gels
• Comparative 2-D gel data analysis
• Western blotting (small format gels)
• HPLC separation of protein mixtures
Mass Spectrometry Laboratory
• Peptide mass fingerprinting
• LC-MS/MS analysis
• Analysis of protein modifications
• Purity analysis of recombinant proteins/synthetic peptides
• Purity analysis of oligonucleotides
Basics of Protein Mass Spectrometry
• Mass spectrometry determines the molecular weight of chemical
compounds by separating molecular ions in a vacuum according to their
mass-to-charge ratio (m/z)
• Ions are generated by induction of either the loss or the gain of a charge
(protonation, deprotonation or electron injection)
• Generated ions can be fragmented in the vacuum, and the resulting subfragments can provide information about the structure of a compound
Ion source
Mass analyzer
Detector
Ion generation
Ion separation
Ion detection
F. Lottspeich and H. Zorbas, Bioanalytik 1998, Spektrum Akad. Verlag
Mass Spectrometry Instrumentation at the GRI
1. Bruker Biflex III MALDI-TOF mass spectrometer
• mid fmole protein/peptide analysis
• protein identification using peptide mass fingerprinting
• oligonucleotide mass/purity analysis
• biomarker analysis
2. Finnigan LCQ Deca XP Max ESI mass spectrometer
• coupled to Dionex Ultimate nanoflow 2-D HPLC
• low fmole peptide analysis
• protein identification using LC-MS/MS peptide sequencing
3. PE Sciex API 3000 ESI mass spectrometer
• low pmole/high fmole peptide/metabolite analysis
• identification of post-translational modifications
• peptide and metabolite quantitation studies
Protein Identification by Mass Spectrometry
1. Peptide Mass Fingerprinting
• protease digestion of protein spots/bands
• peptide extraction
• sample spotting on target plate
• mass measurement of peptide ions by MALDI-TOF MS or LC-MS
• data base search using generated mass list
• protein identification based on ≥ 4 matched peptide masses
2. Peptide Sequencing
• protease digestion of protein spots/bands
• peptide extraction
• RP-LC separation of peptides
• mass measurement and fragmentation analysis of peptide ions
• data base search using parent mass and fragment mass data
• protein identification based on ≥ 2 matched peptides
Intens. [a.u.]
Peptide Mass Fingerprinting
x10 4
1091.620
Sample:
in-gel digested human EF-2
1.5
1799.879
1347.669
1.0
2143.156
1615.722
890.612
1214.658
0.5
1504.667
1978.039
2460.281
2801.340
0.0
1000
1500
2000
2500
3000
m /z
Peptide Mass Fingerprinting Result
Peptide Mass Fingerprinting Result
Tandem Mass Spectrometry (MS/MS) Analysis
eluting peptide
mass analysis
precursor ion
fragmentation
fragment mass analysis
Tandem Mass Spectrometry (MS/MS) Analysis
eluting peptide
mass analysis
precursor ion
T E S T P E P T I D E+
b-ions
b1
b2
b3
b4
b5
b6
b7
b8
b9
b10
b11
T+
TE+
TES+
TEST+
TESTP+
TESTPE+
TESTPEP+
TESTPEPT+
TESTPEPTI+
TESTPEPTID+
TESTPEPTIDE+ - H2O
fragmentation
fragment mass analysis
T E S T + P E P T I D E+
TESTPEPTIDE+
ESTPEPTIDE+
STPEPTIDE+
TPEPTIDE+
PEPTIDE+
EPTIDE+
PTIDE+
TIDE+
IDE+
DE+
E+
y11
y10
y9
y8
y7
y6
y5
y4
y3
y2
y1
y-ions
LC-MS/MS Analysis of Protein Digests
NL:
100
1.21E8
100
Relative Abundance
Relative Abundance
80
60
40
Base Peak
Y12
1299
NL
5.29E6
Base peak
Y6
689
B6/Y132+
706
Y4
475
Y122+
650
B4
493
B3
380
Y10
1087
Y7
803
Y5
588
Y8
902
B9
990
B10
1102
B11
1204
B12
1317
Y13
1412
Y11
1202
0
400
800
1200
1600
m/z
20
0
10
15
20
25
30
35
40
45
50
55
60
Time (min)
Base peak chromatogram of the LC-MS/MS analysis of a protein digest from a silver stained 2D gel spot, the
insert showing the MS/MS spectrum for the actin peptide SYELPDGQVITIGNER as identified by SEQUEST
LC-MS/MS Analysis of Protein Digests
I/L
Relative Abundance
100
T
I/L
V
QG
D
P
I/L
Y12
1299
NL
5.29E6
Base peak
Y6
689
Y4
475
Y10
1087
Y122+
650
B4
493
B3
380
B10
1102
B6/Y132+
706
Y7
803
Y5
588
Y8
902
B9
990
B11
1204
B12
1317
Y11
1202
Y13
1412
0
400
800
1200
m/z
Peptide sequence: SYELPDGQVITIGNER
1600
LC-MS/MS Analysis Result
LC-MS/MS Analysis Result
Frequently Asked Questions
1. How much protein do you need?
Short Answer: At least 1 pmol
Factors:
Long Answer: It depends ...
- protein staining
- protein sequence
- protein size
- potential post-translational modifications
- presence of the protein sequence in the database
2. When can I get the results?
Short Answer: In 1-2 weeks
Factors:
Long Answer: It depends ...
- type of requested analysis
- amount of protein sample
- protein sequence
- protein size
- potential post-translational modifications
- presence of the protein sequence in the database
Frequently Asked Questions
3. I saw a dark band/spot on the gel. Why did we get no results?
1
2
3
4
5
6
7
8
Loading (100 ng protein/lane):
1+2
Ovalbumin (Chicken)
3+4
Myoglobin (Horse)
5+6
Cytochrome C (Horse)
7+8
Serum albumin (Bovine)
Ovalbumin ~ 45 kDa
Myoglobin ~ 17 kDa
Cytochrome C ~ 13 kDa
Serum albumin ~ 66 kDa
100 ng ~ 2.2 pmol
100 ng ~ 5.9 pmol
100 ng ~ 7.9 pmol
100 ng ~ 1.5 pmol
The Limitations
1. Protein Size
Small proteins ( 10 kDa) or large proteins ( 150 kDa) are more
challenging to digest and analyze because they generate few
peptides (small proteins) or show increased resistance to proteases
(large proteins).
2. Protein Sequence
Proteins are typically digested using trypsin (K/R cleavage); the
distribution of these AA dictates the size and the detectability of the
generated peptides.
3. Post-translational Modifications
Glycosylated proteins show high resistance to proteases; certain
post-translational modifications (e.g. phosphorylation) decrease the
detectability of the modified peptide using the standard protein mass
spectrometry techniques.
4. Protein Sequence Databases
The database search algorithms compare the generated spectra with
theoretical digests of proteins in protein sequence databases; the
positive identification of the analyzed protein depends on the
presence of its sequence in those databases.
The Big No-No’s
1. Detergents
Detergents used for extraction and purification of proteins, when not
completely removed, can cause signal suppression and decreased
detectability of peptides in the mass spectrometry analysis
2. Contaminants
In-gel digests of low abundance samples are very sensitive to the
presence of contaminants, particularly contaminating proteins. The
handling of samples/gels with gloves is absolutely necessary and the
use of designated equipment for specific separation and staining
protocols is highly recommended.
3. Formaldehyde or Glutaraldehyde Fixation in Silver Staining
While increasing the staining sensitivity, these fixation steps result in a
covalent modification and cross-linking of proteins, which can result in
decreased digestion efficiency.
Contact Information
Laboratory Address
Proteomics Laboratory
Department of Genome Science (ML 0505)
Genome Research Institute
University of Cincinnati
Building B, Room 131
2180 East Galbraith Road
Cincinnati, Ohio 45237
Tel: 513/558-8950
Fax: 513/558-5061
Email: [email protected]
Staff Members
• Detlef Schumann
• Wendy Dominick
• Michael Wyder
• Margaret Minges